Despite intensive study by developmental biologists up through the 1960s, spiders were forgotten in the following 30 years and little modern work on their development appeared in the literature. Recently, spider embryos have been rediscovered by developmental biologists. Molecular data from the past 9 years suggest that spider embryos use many of the same mechanisms that vertebrate embryos use to establish the body plan. The spider embryo may thus be useful in understanding the developmental mechanisms involved in early development of human embryos. The research proposed in this application will fill a gap in knowledge about a key stage in physical assembly of the embryo: gastrulation. Gastrulation is a process of cell migration by which the egg is reorganized to generate axes of body symmetry and internal organs. In spiders, a 'canonical model'of the gastrulation process was formulated in the 1960s based on analysis of fixed embryos. However, data from the Principal Investigator's lab on living embryos show much deviation from this model. The goal of this proposal is to formulate a new model of spider gastrulation by linking molecular and cellular events with live imaging of development. (1) Mechanism of internalization and cell fates. Cells will be characterized by cadherin and tubulin antibody staining to determine if cells at the blastopore and/or the periphery ingress by epithelial-to- mesenchymal transition. The germ layer fate of both populations of ingressing cells will be determined by tracing fluorescently labeled cells in living embryos and by in situ hybridization for molecular markers of endoderm and mesoderm. (2) Mechanism of elongation of the embryo. Most arthropod embryos elongate by action of a posterior growth zone, but it is not known how the growth zone functions. Cell labeling, tubulin antibody staining, and phospho-Histone H3 antibody staining will be used to distinguish between continued cell ingression, cell rearrangement, cell shape change, and mitosis as mechanisms of growth in the posterior growth zone. (3) Function of the cellular organizer in establishment of the body plan. The hypothesis that the cumulus cells specify the dorsal-ventral axis via dpp expression will be tested and their role examined directly, by removal and transplantation during early, middle, and late gastrulation. Developmental changes will be categorized by both loss and gain of anatomical features and byexpression of dpp and pMAD, the latter a response to signaling by the cumulus. This work will provide original research projects that will train undergraduate students in the process of experimental biology and allow them to present their work at professional meetings. PUBLIC HEALTH RELEVANCE: Gastrulation is a fundamental process of embryonic development that establishes the organization of the body and starts formation of internal organs. This proposal seeks to understand cellular and molecular aspects of spider gastrulation. Because spider embryos are experimentally accessible and rely on cell-cell signaling, just as human embryos do, understanding spider gastrulation will enhance our understanding of early human development.